Interferon antagonist factors encoded by viruses have recently emerged as major virulence factors required for viral replication and pathogenicity in the host. In the case of influenza virus, evasion of the type I interferon system is mainly mediated by the viral non-structural protein 1 (NS1). In this subproject of the PO1 application, we will explore the role of the NS 1 proteins of two highly lethal influenza viruses, the 1918 virus and the influenza A/HongKong/97 virus, in their ability to inhibit the type I interferon system in human, mouse and avian cells. In collaboration with Terry Tumpey and Michael Katze we will also study their contribution to virulence in mouse, avian and monkey models of viral infection. We will determine which specific amino acid sequences in the NS1 proteins of these two influenza viruses are responsible for host specific differences in their ability to evade type I interferon responses and therefore play a critical role in the replication ability of the viruses and their pathogenicity in different hosts. The structural basis for these effects will be investigated in collaboration with Ian Wilson. In addition, we will investigate the antiviral effects of several cellular genes the expression of which is regulated by the NS1 protein, as previously determined by microarray analysis. These experiments will determine the role of known cellular antiviral genes in preventing influenza virus replication, and they will also explore novel host pathways that may be involved in antiviral innate immunity. Finally, we will complete our proposed characterization of the role of individual 1918 viral genes in virus pathogenicity by investigating the phenotypic characteristics of recombinant viruses bearing the M gene of the 1918 virus. These studies will determine whether the M gene encoded products (M1 and M2) significantly contribute to the unusual high lethality of the 1918 virus. The contribution of the M1 protein of 1918 virus in modulating virus particle morphology will also be investigated. By combining the data obtained in this project with the data obtained in projects 3 and 4 we will generate a complete functional characterization of the eight 1918 influenza virus genes and we will determine their specific contributions to the unusual high lethality in humans of this pathogenic strain of influenza A virus.